This proposal will establish a novel and cost effective method for screening at risk populations for active Hepatitis C Virus (HCV) infection by electrically detecting individual protein biomarkers. Our novel method blends the single-molecule sensitivity of a nanopore with high-affinity reagents designed to target only specific biomarkers of interest. Achievable characteristics of our portable device include: high assay sensitivity (<5 IU/ml) and accuracy (99.9% confidence), ease of use (in-line automation), rapid time to results (<10 min, sample-in to results-out), and low cost (~2$/test). This proposal focuses on detecting the core antigen of HCV (HCVcAg), since it is a well-studied and short-lived biomarker that can be quantitated for the diagnosis of both acute and chronic infections. To achieve HCV detection, we use bi-functional fusion molecules in which one end of the fusion captures the target HCVcAg in solution while the other end anchors the fusion/biomarker complex to a unique location on a dsDNA scaffold. When each scaffold/fusion passes through the pore, the occupancy status of the fusion is detected because the event depth and/or duration values are larger for the larger molecule (i.e., when the biomarker is bound), providing a simple yes/no criterion for every event. Moreover, our assays are designed so that all other background molecules (e.g., from serum) produce events distinguishable from the scaffold/fusion complexes. The software filters out background events, thereby allowing for quantitation of the HCVcAg-bound scaffolds. This proposal has three aims: Aim 1 (4 months): Create the dsDNA/fusion molecule and biochemically show that it binds the HCVcAg biomarker in solution. Fusion molecules comprise a dsDNA-binding Protein Nucleic Acid domain and an HCVcAg specific DNA aptamer. Formation of dsDNA/fusion/HCVcAg will be confirmed using gel shift assays. Aim 2 (4 months): Using the DNA/fusion reagent and nanopore technology, electrically detect the presence of HCVcAg biomarkers in solution. Existing mathematical tools will assess the statistical significance of detection. Aim 3 (4 months): Titrate HCVcAg to establish the limit of biomarker detection, and perform experiments in the presence of background protein and diluted serum. This aim investigates the effects of background on detection, and helps assess if reagent redesign is needed due to non-specific interactions with the dsDNA/fusion.